Acyl carrier protein (ACP) is a small acidic protein (8,800 Da) responsible for acyl group activation in fatty acid biosynthesis. The gene encoding ACP (acpP) has been cloned and overexpressed (Rawlings, M. and Cronan, J. E., Jr. (1992) J. Biol. Chem., 267, 5751-5754; Jones, A. L., et al. (1993) Biochem. Soc. Trans., 21, 202S) and the solution structure of ACP has been solved by NMR spectroscopy (Holak, T. et al. (1988) Eur. J. Biochem. 175:9-15). Homologs of E. coli ACP exist throughout nature in two forms; either as an integral domain of a much larger multifunctional enzyme (type I) or as a discrete protein capable of associating with several other enzymes constituting a multienzyme synthase complex (type II). In these two forms, ACPs play central roles in a broad range of other biosynthetic pathways that depend on iterative acyl transfer steps, including polyketide (Shen, B., et al. (1992) J. Bacteriol. 174:3818-3821), non-ribosomal peptide (Baldwin, J. E., et al. (1991) J. Antibiot. 44:241-247), and depsipeptide biosynthesis (Rusnak, F., et al. (1991) Biochemistry 30:2916-2927) as well as in the transacylation of oligosaccharides (Geiger, O., et al. (1991) J. Bacteriol. 173:2872-2878) and proteins (Issartel, J. P., et al. (1991) Nature 351:759-761).
A definitive feature of ACP is the 4xe2x80x2-phosphopantetheine (4xe2x80x2-PP) prosthetic group (Majerus, P. W. et al. (1965) Proc. Natl. Acad. Sci. USA 53:410-417). 4xe2x80x2-PP is attached through a phosphodiester linkage to a conserved serine residue found in all ACPs. Acyl groups of the many substrates recognized by type I and type II ACPs are activated for acyl transfer through a thioester linkage to the terminal cysteamine thiol of the 4xe2x80x2-PP moiety. The xcex2-alanyl and pantothenate portions of the 4xe2x80x2-PP structure are believed to serve as a tether between the phosphodiester-ACP linkage and the terminal thioester, suggesting that 4xe2x80x2-PP may function as a swinging arm, shuttling growing acyl chains between various active sites, e.g. as in the sequential addition of 11 amino acids by the 800 kDa cyclosporin synthetase (Lawen, A. and Zocher, R. (1990) J. Biol. Chem. 265:11355-11360).
Holo-ACP synthase (holo-ACPS) transfers the 4xe2x80x2-PP moiety from Coenzyme A (CoA) to Ser-36 of apo-ACP to produce holo-ACP and 3xe2x80x2,5xe2x80x2-ADP in a Mg2+ dependent reaction. The (acyl carrier synthase protein) ACPS from E. coli was partially purified 780-fold from crude extracts (Elovson, J. and Vagelos, P. R. (1968) J. Biol. Chem. 243:3603-3611), and the ACPS from spinach has been partially purified (Elhussein, S. A., et al. (1988) Biochem. J. 252:39-45), but remarkably little has been shown about the mechanism or specificity of this post-translational phosphopantetheinylation process. A mutant of E. coli conditionally defective in the synthesis of holo-ACP has been identified and the mutant phenotype attributed to an altered holo-ACP synthase activity (Polacco, M. L. and Cronan, J. E., Jr. (1981) J. Biol. Chem. 256:5750-5754).
This invention pertains to isolated and purified natural and recombinant phosphopantetheinyl transferases, e.g., acyl carrier protein synthases (ACPSs), from eukaryotes, prokaryotes, or plants. Also within the scope of the invention are active fragments of phosphopantetheinyl transferases, modified phosphopantetheinyl transferases, and modified active fragments of phosphopantetheinyl transferases. These forms of phosphopantetheinyl transferase are preferably purified to at least about 60% purity, more preferably to at least about 70% purity, more preferably to at least about 80% purity, more preferably to at least about 90% purity and even more preferably to at least about 95% purity. The phosphopantetheinyl transferase of the invention can be used for in vitro phosphopantetheinylation of substrates, such as acyl carrier proteins (ACPs), which have, for example, been produced by overexpression in a host cell. Kits including the phosphopantetheinyl transferase described herein are also within the scope of the invention.
The invention also provides host cells modified to express at least one nucleic acid encoding at least one phosphopantetheinyl transferase or active fragment thereof. In one embodiment, the host cells of the invention are further modified to express at least one nucleic acid encoding at least one substrate of a phosphopantetheinyl transferase. Such host cells may further express nucleic acids encoding other components associated with the ACP. Modified host cells of the invention can be used for the production of antibiotics or other compounds whose synthesis requires an ACP.